An oligonucleotide binds to a target nucleic acid by forming hydrogen bonds between bases in the target and the oligonucleotide. Common B DNA has conventional adenine-thymine (A-T), and guanine-cytosine (G-C) Watson and Crick base pairs with two and three hydrogen bonds, respectively. Conventional hybridization technology is based upon the capability of sequence-specific DNA or RNA probes to bind to a target nucleic acid via Watson-Crick hydrogen bonds. However, other types of hydrogen bonding patterns are known wherein some atoms of a base which are not involved in Watson-Crick base pairing can form hydrogen bonds to another nucleotide. For example, thymine (T) can bind to an A-T Watson-Crick base pair via hydrogen bonds to the adenine, thereby forming a T-AT base triad. Hoogsteen (1959, Acta Crystallography 12: 822) first described the alternate hydrogen bonds present in T-AT and C-GC base triads. More recently, G-TA base triads, wherein guanine can hydrogen bond with a central thymine, have been observed(Griffin et al., 1989, Science 245: 967-971). If an oligonucleotide could bind to a target with both Watson-Crick and alternate hydrogen bonds an extremely stable complex would form that would have a variety of in vivo and in vitro utilities. However, to date there has been no disclosure of an oligonucleotide with the necessary structural features to achieve stable target binding with both Watson-Crick and alternate hydrogen bonds.
Oligonucleotides have been observed to bind by non-Watson-Crick hydrogen bonding in vitro. For example, Cooney et al., 1988, Science 241:456 disclose a 27-base single-stranded oligonucleotide which bound to a double-stranded nucleic acid via non-Watson-Crick hydrogen bonds. However, triple-stranded complexes of this type are not very stable, because the oligonucleotide is bound to its target only with less stable alternate hydrogen bonds, i.e., without any Watson-Crick bonds.
Oligonucleotides have been used for a variety of utilities. For example, oligonucleotides can be used as probes for target nucleic acids that are immobilized onto a filter or membrane, or are present in tissues. Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, N.Y.) provide a detailed review of hybridization techniques.
Furthermore, there has been great interest recently in developing oligonucleotides as regulators of cellular nucleic acid biological function. This interest arises from observations on naturally occurring complementary, or antisense, RNA used by some cells to control protein expression. However, the development of oligonucleotides for in vivo regulation of biological processes has been hampered by several long-standing problems, including the low binding stability and nuclease sensitivity of linear oligonucleotides.
For example, transcription of the human c-myc gene has been inhibited in a cell free, in vitro assay system by a 27-base linear oligonucleotide designed to bind to the c-myc promoter. Inhibition was only observed using a carefully controlled in vitro assay system wherein lower than physiological temperatures were employed, and many cellular enzymes had been removed or inactivated. These conditions were necessary because linear oligonucleotides bind with low affinity and are highly susceptible to enzymes which degrade linear pieces of DNA (Cooney et al.). Splicing of a pre-mRNA transcript essential for Herpes Simplex virus replication has also been inhibited with a linear oligonucleotide which was complementary to an acceptor splice junction. In this instance, a methylphosphonate linkage was employed in the linear oligonucleotide to increase its nuclease resistance. Addition of this chemically-modified oligonucleotide to the growth medium caused reduction in protein synthesis and growth of uninfected cells, most likely because of toxicity problems at high concentrations (Smith et al., 1986, Proc. Natl. Acad. Sci. USA 83: 2787-2791).
In another example, linear oligonucleotides were used to inhibit human immunodeficiency virus replication in cultured cells. Linear oligonucleotides complementary to sites within or near the terminal repeats of the retrovirus genome and within sites complementary to certain splice junctions were most effective in blocking viral replication. However, these experiments required large amounts of the linear oligonucleotides before an effect was obtained, presumably because of the low binding stability and vulnerability of these linear oligonucleotides to nucleases (Goodchild et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5507-5511).
Accordingly, oligonucleotides that are useful as regulators of biological processes preferably possess certain properties. First, the oligonucleotide should bind strongly enough to its complementary target nucleic acid to have the desired regulatory effect. Second, it is generally desirable that the oligonucleotide and its target be sequence specific. Third, the oligonucleotide should have a sufficient half-life under in vivo conditions for it to be able to accomplish its desired regulatory action in the cell. Hence, the oligonucleotide should be resistant to enzymes that degrade nucleic acids, e.g. nucleases. Fourth, the oligonucleotide should be able to bind to single- and double-stranded targets.
While linear oligonucleotides may satisfy the requirement for sequence specificity, linear oligonucleotides are sensitive to nucleases and generally require chemical modification to increase biological half-life. Such modifications increase the cost of making an oligonucleotide and may present toxicity problems. Furthermore, linear oligonucleotides bind to form a two-stranded complex like those present in cellular nucleic acids. Consequently, cellular enzymes can readily manipulate and dissociate a linear oligonucleotide bound in a double-stranded complex with target. The low binding strength and nuclease sensitivity of linear oligonucleotides can thus necessitate administration of high concentrations of oligonucleotide, in turn making such administration toxic or costly. Moreover, while linear oligonucleotides can bind to a double-stranded target via alternate hydrogen bonds (e.g. Hoogsteen binding), linear oligonucleotides cannot readily dissociate a double-stranded target to replace one strand and thereby form a more stable Watson-Crick bonding pattern.
Furthermore, increased binding strength increases the effectiveness of a regulatory oligonucleotide. Therefore, an oligonucleotide with high binding affinity can be used at lower dosages. Lower dosages decrease costs and reduce the likelihood that a chemically-modified oligonucleotide will be toxic. Therefore, high oligonucleotide binding affinity for target is a highly desirable trait.
Accordingly, the present invention provides single-stranded circular oligonucleotides which, by nature of the circularity of the oligonucleotide and the domains present on the oligonucleotide, are nuclease resistant and bind with strong affinity and high selectivity to their targeted nucleic acids. Moreover, the present circular oligonucleotides can dissociate and bind to a double-stranded target without prior denaturation of that target.
Some types of single-stranded circles of DNA or RNA are known. For example, the structures of some naturally occurring viral and bacteriophage genomes are single-stranded circular nucleic acids. Single-stranded circles of DNA have been studied by Erie et al. (1987, Biochemistry 26:7150-7159 and 1989, Biochemistry 28: 268-273). However, none of these circular molecules are designed to bind a target nucleic acid. Hence, the present invention represents an innovation characterized by a substantial improvement relative to the prior art since the subject circular oligonucleotides exhibit high specificity, low or no toxicity and more resistance to nucleases than linear oligonucleotides, while binding to single- or double-stranded target nucleic acids more strongly than conventional linear oligonucleotides.